Organ transplantation is a very valuable and radical therapy. Such organs as the kidney, liver and heart have been transplanted to treat some organ failure. Transplantation can be classified into allotransplantation (transplantation between a donor and a recipient of the same species) and xenotransplantation (that between those of different species). Such transplantation has both advantages and disadvantages. Allotransplantation is superior to the xenotransplantation because of less rejection, but the former is inferior to the latter because of shortage of human donors. Recently, animal-to-man organ transplantation (xenotransplantation) has been studied mainly in European countries and the United States. Apes may be desirable donors because of close relation to human beings, but the use of their organs may be infeasible because of the shortage of these animals and their high intelligence. However, farm animals, particularly pigs, have advantages of their organ sizes and shapes similar to those of man, abundant supply due to mass rearing and established basic technology. Consequently, the pig-to-man organ transplantation has mainly been studied. Xenotransplantation between closer species (e.g., baboon-to-man) and that between more distant species (e.g., pig-to-man) are called concordant and discordant xenotransplantation, respectively.
In pig-to-man discordant xenotransplantation, the xenograft will be rejected within minutes. Such a phenomenon is called hyperacute rejection, which is initiated by the reaction between natural antibodies in the human serum and the xenoantigens in the porcine xenografts (organs, tissues, cells, etc.), resulting in complement-dependent cell injury (complement reaction).
Gal α 1,3Gal (referred to as α-Gal antigen hereafter) at non-reducing terminals of sugar chains (sugar protein and sugar lipid) on the porcine cells has been regarded as one of the significant xenoantigens. The α-Gal antigen is synthesized upon addition of α-1,3-galactose to N-acetyl-lactosamine by α-1,3-galactosyltransferase (hereafter referred to as α-1,3GT) (FIG. 1).
Human tissue do not expresse α-Gal antigen. Of mammals, Old World monkeys (inhabiting in Asia and Africa), anthropoids (the chimpanzee, gorilla and orangutan) and man, taxonomically stemmed out 20-30 million years ago, are carrying the frame-shifted α-1,3GT pseudogene (J. Biol. Chem., 265: 7055-7061 (1990)) but producing the natural antibodies against the α-Gal antigens. It has been reported that anti-α-Gal antibody comprises as much as 1% of human IgG (Blood, 82: 2485-2493 (1993)).
Even if hyperacute rejection is overcome, such graft failure as endothelial swelling, ischemia and thrombosis will occur. Histologically, coagulation of platelets, deposit of fibrin, activation of endothelia and invasion of activated macrophages and NK cells will occur. Such a phenomenon is called acute vascular rejection (AVR) or delayed xenograft rejection (DXR). Xenoreactive antigens, complement, macrophages, NK cells, neutrophils and platelets may be the factors implicated in initiation of AVR.
The method preventing hyperacute rejection includes those applicable to the recipients and those applicable to the donors. An example of the former is adsorption of the natural antibodies onto immunologically-prepared columns, and those of the latter are expression of the recipients' complement inhibitors and reduction of the α-Gal antigens. Because the natural antibodies can hardly be removed from the recipients, the treatments of the donors are generally adopted. Because the xenoantigens may cause AVR, reduction in the α-Gal antigens may be effective on retarding or preventing AVR.
The methods to reduce the donors' α-Gal antigens are as follows:    (1) Reduction of α-Gal antigen by digestion of α-galactosyl bond at a sugar-chain terminal with α-galactosidase
Although α-galactosidase from coffee beans is known, the enzyme failed to physiologically digest the α-Gal antigen because of its optimum pH ranging between 6 and 6.5.    (2) knock out (KO) of α-1,3GT gene responsible for α-Gal antigen production
Although α-1,3GT gene-KO mice were developed (Transplantation, 61: 13-19 (1996)), they suffered from cataract. Since pigs are expressing more α-Gal antigens than mice, α-1,3GT gene-KO pigs may cause severer adverse effects.
Furthermore, KO-pig development itself has been infeasible, because no porcine embryonic stem (EC) cell has been established.    (3) competitive prevention of α-1,3GT gene by introducing genes of other sugar transferases sharing the same substrate (N-acetyllactosamine)
α-Fucosyltransferase (α-1,2FT) gene was introduced to convert galactose of the α-Gal antigens (Gal α 1,3Gal) synthesized by α-1,3GT to fucose or an H blood-type substance (WO095/34202A1). Such methods may reduce the α-Gal antigens, because galactose at the non-reducing sugar-chain terminals is replaced with other sugars. However, the overall reduction of the α-Gal antigens is negligible, unless expression of the introduced gene exceeds that of the endogenous α-1,3GT gene. Moreover, such methods can not reduce the number of sugar chains or their branches themselves in contrast with N-acetylglucosaminyltransferase III described later.    (4) Forced expression of N-acetylglucosaminyltransferase III (β-D-mannoside β-1,4 N-acetylglucosaminyltransferase III, EC 2.4.1.144: referred to as GnT-III in the following) to prevent N-linked sugar chain from branching
Forced expression of GnT-III results in prevention of sugar-chain branching and α-Gal antigen production. The mechanisms of sugar-chain branching and its prevention by GnT-III are illustrated in FIG. 2.
Generally, the sugar chains can be classified into N-, O- and lipid-linked ones. Each sugar chain has a core structure, from which the sugar chain extends to its terminal. Such extension will end, if α-1,3 galactose is added. Similarly, α-1,2FT will stop the sugar-chain extension from a core structure of N-acetyllactosamine. Consequently, such transferases are antagonistic to α-1,3GT (see FIG. 1). However, GnT-III relates to formation of the core structure of N-linked sugar chains like other N-acetylglucosaminyl (GlcNAc)transferases and is not antagonistic to other sugar transferases.
N-linked sugar chains can be classified into two-, three- and four-branched ones. As FIG. 2 shows, GnT-IV and GnT-V catalyze introduction of a sugar branch to a precursor two-branched sugar chain, resulting in three- and four-branched ones. GnT-I and GnT-II catalyze introduction of a branch to a precursor of a core N-linked sugar chain, resulting in two-branched one. If GlcNAc is added to β-mannose (β-Man) of the core structure by GnT-III, no sugar chain can extend at all. Such GlcNAc is, therefore, distinguished from other GlcNAc and called bisecting GlcNAc. If bisecting GlcNAc is added to a two-branched sugar chain, neither GnT-IV nor GnT-V will use it as their substrate nor synthesize three- or four-branched sugar chains. Arrows with squares in FIG. 2 indicate such inhibition of the enzymatic reactions.
The two-branched sugar chain with bisecting GlcNAc inhibits β-galactosyl and α-galactosyl reactions as well as α-Gal antigen synthesis (Glycobiol., 6: 691-694 (1996)). In summary, forced expression of GnT-III reduces the sugar-chain branching and the α-Gal antigen production of not only each sugar chain but also the entire cell.
As for GnT-III-introduced transgenic animals, transgenic mice were generated by a transgene comprising rat GnT-III cDNA, β-actin promoter and cytomegalovirus enhancer (Transplant. Proc., 29: 895-896 (1997)). Their hearts, lungs and livers expressed more GnT-III and less α-Gal antigens than did those in normal mice. The normal mice, however, expressed much GnT-III (3,400 p mol/h/mg protein) in their kidneys. Although the transgenic mice expressed more GnT-III (4,800 p mol/h/mg protein) in their kidneys, they failed to reduce the α-Gal antigens in their kidneys. Namely, the transgenic mice carrying the rat GnT-III gene failed to reduce the α-Gal antigens in their organs, especially their kidneys.
No GnT-III-introduced transgenic pig has ever been generated. Viviparous mammals except marsupials and anthropoids express the α-Gal antigens on their cell membranes. Particularly, porcine organs express much α-Gal antigens. For example, porcine kidneys express 500- to 1,000-times more α-Gal antigens than do mouse kidneys. The α-Gal antigens may be indispensable for development and survival of pigs. They may mask the bacterial receptors and prevent infection (Cell Engineering, 19: 830-834 (2000)). As described later, no normal pig organ such as the kidney expresses GnT-III at all. It has been unknown whether or not artificial expression of GnT-III on porcine organs, tissues or cells affects porcine development and survival, and whether the artificial expression of GnT-III reduces the α-Gal antigens. Of promoters used to construct transgenes, it is known that some can not necessarily induce expression of objective structure genes in pigs, even if they effectively work in mice (e.g., Theriogenol. 51: 422 (1999)). Consequently, no gene promoter effectively inducing expression of the human GnT-III in pigs has been known.
Particularly, the following have been unknown, (1) whether liveborn transgenic piglings can be generated from embryos injected with GnT-III gene (especially, the human GnT-III gene), (2) even if the transgenic pigs carrying the human GnT-III gene are generated, whether their organs can express GnT-III and reduce the α-Gal antigens, (3) whether liveborn transgenic pigs carrying both the GnT-III gene and one or more transgenes other than GnT-III in their individual bodies can be generated, (4) whether their organs can express GnT-III, reduce the α-Gal antigens and express one or more transgenes other than GnT-III gene, and (5) if one or more transgenes expressing along with the sugar-chain remodeling GnT-III gene is (are) a sugar protein(s) (e.g., human DAF), whether expression of GnT-III will injure expression of such sugar protein(s) (e.g., the human DAF).
Since hyperacute rejection takes place in local cells (e.g., vascular endothelial cells), such transgenic mammals with reduced N-linked sugar-chain branches and reduced α-Gal antigens in local cells have been desired.
Since complement-dependent cell injury (complement reaction) initiates hyperacute rejection, generation of transgenic mammals expressing complement inhibitor (human complement inhibitor in case of human recipients) and less α-Gal antigens have been desired to suppress the complement reaction of the transplantation recipient.